Peptide 1 gel alone and MTH comprising 16 mM tofacitinib, 5% (v/v) DMSO and 1% (w/v) peptide 1 was prepared in syringes equipped with 27G needles. MTH and gel alone were syringe-injected into Corning® Transwell® microporous membrane inserts (8 µm) for transferring 12 µL and 50 µL gels in each group. The inserts were placed onto a 24-well plate seeded with murine bone marrow-derived dendritic cells (BMDCs). The cells were seeded in 24-well plate (2X105 cells/mL per plate) on Day 0, with the addition of the MTH and Peptide 1 gel alone onto transwell inserts on Day 6. BMDCs were stimulated with LPS (Enzo, Lot # 02261813) at 200 ng/mL on Day 7 for overnight, collected on Day 8, and stained with anti-CD11c-FITC (N418, eBioScience), anti-CD80-APC (16-10A1, BioLegend), anti-CD86-eF450 (GL1, eBioscience), and anti-MHCII-eF710 (M5/114.15.2, eBioScience) antibodies. The expression levels of these maturation markers were measured on a BD LSRII flow cytometer and the data were analyzed using FlowJo software (version 10.5.3, TreeStar) by gating on CD11c+ single cells.
Anti cd11c fitc n418
Manufactured by Thermo Fisher Scientific
Anti-CD11c-FITC (N418) is a fluorescently labeled antibody that specifically binds to the CD11c protein, which is a marker commonly expressed on dendritic cells. This product can be used for the identification and/or isolation of cells expressing CD11c in flow cytometry applications.
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Lab products found in correlation
2 protocols using anti cd11c fitc n418
1
Peptide-Based Immunomodulatory Hydrogel
2
Isolation of Thymocytes, Tregs, Thymic DCs, and TECs
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For analysis of thymocyte and Treg development thymii and peripheral lymphoid organs were mechanically dissociated into PBS with 2% FBS and 2 mM EDTA, pH 7.4, using frosted glass slides. Cell suspensions were passed through 70 μm filters and washed prior to staining. For analysis of thymic DC and epithelial cells (TEC), thymii were dissected into 5 mL complete RPMI supplemented with Collagenase D (120 Mandl U/mL) and DNase I (3 U/ml, both from Roche Applied Sciences, Indianapolis, IN) and diced into ~20 pieces and placed in a 37°C incubator for 30 minutes. Pieces were further homogenized by pipetting and the addition of EDTA (20 mM final concentration) which was immediately diluted 10-fold with buffer and the suspension filtered through 70 μm mesh. Thymic DC were magnetically enriched by labeling with anti-CD11C FITC (N418, eBioscience, San Diego, CA) and secondarily with anti-FITC microbeads (Miltenyi Biotec, Auburn, CA). Thymic epithelial cells were enriched by magnetic depletion of leukocytes using biotinylated anti-CD45 antibody (30F11, eBioscience) and streptavidin-conjugated microbeads (Miltenyi Biotec).
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